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The inhibitory effects of silver nanoparticles, silver ions,
and silver chloride colloids on microbial growth
Okkyoung Choi
a
, Kathy Kanjun Deng
b
, Nam-Jung Kim
c
, Louis Ross Jr.
d
, Rao Y. Surampalli
e
,
Zhiqiang Hu
a,
a
Department of Civil and Environmental Engineering, University of Missouri, E2509 Lafferre Hall, Columbia, MO 65211, USA
b
School of Engineering, Rice University, USA
c
Department of Mechanical and Aerospace Engineering, University of Missouri, USA
d
Electron Microscopy Core Facility, University of Missouri, USA
e
Region 7 Office, US Environmental Protection Agency, Kansas City, KS, USA
a r t i c l e
i n f o
Article history:
Received 9 October 2007
Received in revised form
22 February 2008
Accepted 26 February 2008
Available online 4 March 2008
Keywords:
Silver nanoparticles
Silver ion
AgCl colloid
Microbial growth
Nitrification
Inhibition
a b s t r a c t
Emerging nanomaterials are of great concern to wastewater treatment utilities and the
environment. The inhibitory effects of silver nanoparticles (Ag NPs) and other important Ag
species on microbial growth were evaluated using extant respirometry and an automatic
microtiter fluorescence assay. Using autotrophic nitrifying organisms from a well-controlled
continuously operated bioreactor, Ag NPs (average size ¼ 14
76 nm), Ag
+
ions (AgNO
3
), and
AgCl colloids (average size ¼ 0.25 mm), all at 1 mg/L Ag, inhibited respiration by 86
73%,
42
77%, and 4674%, respectively. Based on a prolonged microtiter assay, at about 0.5 mg/L
Ag, the inhibitions on the growth of Escherichia coli PHL628-gfp by Ag NPs, Ag
+
ions, and AgCl
colloids were 55
78%, 100%, and 6676%, respectively. Cell membrane integrity was not
compromised under the treatment of test Ag species by using a LIVE/DEAD Baclight
TM
bacterial viability assay. However, electron micrographs demonstrated that Ag NPs attached
to the microbial cells, probably causing cell wall pitting. The results suggest that nitrifying
bacteria are especially susceptible to inhibition by Ag NPs, and the accumulation of Ag NPs
could have detrimental effects on the microorganisms in wastewater treatment.
&
2008 Elsevier Ltd. All rights reserved.
1.
Introduction
Nanosilver (silver nanoparticle, Ag NP) materials have a wide
range of applications including spectrally selective coating for
solar energy absorption (
), catalysis in chemical reactions (
),
surface-enhanced Raman scattering for imaging (
), and antimicrobial sterilization (
). Because
of their effective antimicrobial properties and low toxicity
toward mammalian cells, Ag NPs have become one of the
most commonly used nanomaterials in consumer products
(104 out of 502 nanoproducts surveyed) (
). These nanoparticles will likely enter the
sewage pipes and the wastewater treatment plants (WWTPs).
At present, little is known about the adverse effects of Ag NPs
on wastewater treatment and the environment.
It is known, however, that free silver ion (Ag
+
) is highly toxic
to a wide variety of organisms including bacteria. Metal
toxicity to planktonic species such as algae (
)
and bacteria (
) is often governed by the
concentrations of aqueous free metal species (i.e., Ag
+
). The
inhibitory effect of Ag
+
is believed to be due to its sorption to
the negatively charged bacterial cell wall, deactivating
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4 2 ( 2 0 0 8 ) 3 0 6 6 – 3 0 7 4
cellular enzymes, disrupting membrane permeability, and
ultimately leading to cell lysis and death (
). The aqueous concentrations of Ag
+
are typically
low in wastewater treatment systems or in the natural
environment because of its strong complexation with various
ligands such as chloride (K
sp
¼
10
–9.75
), sulfide (K
sp
¼
10
–49
),
thiosulfate, and dissolved organic carbon (
;
). As a result, silver toxicity to microorganisms is
generally not observed.
Nanosilver, a particle of Ag element, is a new class of
material with remarkably different physiochemical character-
istics such as increased optical, electromagnetic and catalytic
properties from the bulk materials (
;
). Nanoparticles with at least one dimension of
100 nm or less have unique physicochemical properties, such
as high catalytic capabilities and ability to generate reactive
oxygen species (ROS) (
) (see recent review
by
). Silver in the form of nanoparticles could be
therefore more reactive with its increased catalytic properties
and become more toxic than the bulk counterpart. Further-
more, toxicity is presumed to be size- and shape-dependent
(
), because small size nanoparticles (e.g.,
o10 nm) (
) may pass
through cell membranes and the accumulation of intracellular
nanoparticles can lead to cell malfunction.
Little work has been done to evaluate the inhibition of
microbial growth by different Ag species, especially Ag NPs in
wastewater treatment systems where such information is
valuable for operation planning and control. Both autotrophic
and heterotrophic microorganisms are important in waste-
water treatment. While heterotrophs are responsible for
organic and nutrient removal, autotrophs are responsible for
nitrification that is considered as the controlling step in
biological nitrogen removal because of the slow growth rate of
nitrifying organisms and their sensitivity to temperature, pH,
dissolved oxygen (DO) concentration, and toxic chemicals
(
). Consequently, the
objective of this research was to evaluate the impact of
important Ag species such as Ag NPs, Ag
+
ions, and AgCl
colloids on heterotrophic and autotrophic growth.
In this research work, Ag NPs and AgCl colloids with larger
sizes were synthesized and characterized by UV–vis spectro-
scopy and electron microscopy. The inhibitory effects on the
autotrophic and heterotrophic growth were determined by a
short-term extant respirometric assay and an automatic
microtiter assay, respectively. Environmental scanning elec-
tron microscopy (ESEM) was applied as a complementary
technique to examine the microbial/nanoparticle interac-
tions. The mode of action of nanosilver toxicity was finally
discussed based on the results of membrane integrity using a
LIVE/DEAD Baclight
TM
bacterial viability kit.
2.
Materials and methods
2.1.
Silver materials
2.1.1.
Silver nanoparticles
Ag NPs were synthesized by reducing silver nitrate with
sodium borohydrate (NaBH
4
) and adding polyvinyl alcohol
(PVA) (Aldrich) as the capping agent to control the growth of
nanocrystals and agglomeration of nanoparticles. To dissolve
PVA, a solution containing 0.06% (wt) PVA was heated to
100 1C and cooled down to room temperature before use.
Silver particles were prepared by rapidly injecting 0.5 mL of
10 mM NaBH
4
into 20 mL PVA solution containing 0.25 mM
silver nitrate at room temperature. After 5 min of stirring, the
reaction mixture was stored at 4 1C before use.
2.1.2.
Silver ions
A silver nitrate standard solution (14 mM, Fisher Scientific)
was used as a source of Ag
+
ions.
2.1.3.
Silver chloride colloids
Aliquots of 100 mg/L AgCl colloids were prepared freshly by
vigorously mixing (700 rpm) 1 mL of 14 mM silver nitrate
standard solution and 1 mL of 28 mM sodium chloride with
18 mL of distilled water. Twice as much sodium chloride as
silver nitrate was added to ensure complete complexation
with no residual Ag
+
ions in the colloidal solution (confirmed
by Ag
+
measurements with an ion-selective electrode).
2.2.
Microbial cultures
2.2.1.
Autotrophic bacteria
The mixed and enriched nitrifying bacteria were cultivated in
a continuously stirred tank reactor (14 L) operated at solids
retention time of 20 d and hydraulic retention time of 1 d
using seed from a local nitrifying activated sludge plant in
Missouri, USA. The reactor was fed with an inorganic medium
containing ammonium (8.3 mM, NH
4
NO
3
) as the sole energy
source and requisite macro- and micronutrients (
). Low
concentrations of anions such as chloride and sulfate were
present in the reactor to minimize their complexation
potential with Ag
+
ions. Sodium carbonate (0.5 M) was
intermittently added to maintain the reactor pH at 7.5
70.1
and fulfilled both carbon and alkalinity requirements. After a
few months of operation, mixed liquor was periodically
withdrawn from the nitrifying reactor for batch respirometric
studies.
2.2.2.
Heterotrophic bacteria
The test heterotrophic bacterium was Escherichia coli PHL628-
gfp, a gift from Dr. Anthony Hay at Cornell University. This
strain tagged with a green fluorescence protein (GFP) was a
derivative of the E. coli K12 that forms biofilms as a
consequence of the over-expression of curli (
). The test strain was grown overnight on a mechanical
shaker (200 rpm) at room temperature in a nutrient-rich
medium (BBL
TM
containing 5 g/L Gelysate
TM
peptone and
3 g/L beef extract, pH 6.9
70.2).
2.3.
Silver species characterization
Aliquots of the prepared Ag NP suspensions were periodically
scanned from 250 to 700 nm to obtain absorption spectra
using a UV–vis spectrophotometer (Cary 50, Varian, CA).
Additional aliquots were used to determine the stability of
the Ag NPs by measuring the concentrations of Ag
+
ions in the
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Ag NP suspensions using a silver ion/sulfide selective
electrode (Denver instrument, Denver, CO).
The sizes of Ag NPs and AgCl colloids were characterized by
an FEI Quanta 600F ESEM (resolution: 3 nm at 30 kV, FEI
Company, OR) equipped with a scanning transmission
electron microscopy (STEM) detector. The Ag NP suspension
was added to standard carbon-coated TEM grid. Images of the
samples were taken at an accelerating voltage of 30 keV.
2.4.
Autotrophic growth determined by extant
respirometry
Autotrophic growth inferred from oxygen uptake rates due to
ammonia oxidation was measured in triplicate using a batch
extant respirometric assay (
). Aliquots (60 mL) of
nitrifying bacteria were collected from the nitrifying reactor.
MOPS[3-(N-morpholino) propanesulfonic acid, pH adjusted to
7.5] at a final concentration of 20 mM was added to maintain
relatively constant pH of 7.5 during ammonium oxidation.
The nitrifying bacterial suspensions were amended with Ag
NPs, Ag
+
ions and AgCl colloids individually at the final
concentration range of 0.1–1 mg/L Ag, filled into the respiro-
metric bottles with no headspace, and then tightly capped.
Every batch respirometric test was accompanied by a positive
control (e.g., untreated nitrifying bacteria only) at room
temperature (25
72 1C). The nitrifying bacterial suspensions
were aerated with pure oxygen gas before aliquots of NH
4
+
-N
(10 mg/L N as NH
4
NO
3
) were injected. Magnetic stirring at ca.
100 rpm was provided in the bottles to ensure complete
mixing. A decrease in the DO level in the respirometric vessel
was measured by a DO probe (YSI model 5300A, Yellow
Springs, OH) and continuously monitored at 4 Hz by an
interfaced personal computer. The degree of inhibition of
autotrophic microbial growth was inferred from the differ-
ence between the measured specific oxygen uptake rate in
the absence and presence of the Ag species (
).
2.5.
Heterotrophic growth determined by an automated
microtiter assay
To evaluate the inhibitory effects of Ag species on hetero-
trophic growth, E. coli PHL628-gfp was grown in nutrient broth
(BBL) at room temperature overnight. For the microtiter
fluorescence assay, aliquots of the fresh medium (190 mL)
were pipetted into eight parallel wells of a 96-well microplate
(i.e., 8 replicates), and aliquots (10 mL) of overnight E. coli
cells were inoculated in each well. Aliquots of the Ag NP
suspension, Ag
+
or AgCl colloidal solution were added
individually to each well to reach predetermined Ag concen-
trations. The cells were exposed to ambient air and mixed
intermittently to support their growth on the plate. A program
was made to incubate the samples with vigorous mixing for
10 s per hour before the fluorescence intensities (535 nm)
excited at 485 nm were recorded automatically every hour for
24 h. The plate was pre-equilibrated at room temperature
(25
72 1C) for 0.5 h and the fluorescence (in relative fluores-
cence unit, RFU) of microbial suspensions was measured with
a fluorescence microreader (VICTOR
3
, PerkinElmer, Shelton,
USA).
The time-dependent microbial growth associated with
organic substrate oxidation in the 96-microwells was simu-
lated using an exponential growth model:
X ¼ X
0
e
m
t
(1)
where X and X
0
are final and initial biomass concentrations,
respectively, as reflected by the fluorescence intensity. The
parameters of the specific microbial growth rate, m, were
determined via least-squares error analysis using the SOLVER
routine in Microsoft Excel.
2.6.
Microbial/nanoparticle interaction
The microbial/nanoparticle interaction was visualized using
the FEI Quanta 600F SEM in the environmental (ESEM) mode
that allows organic samples to be examined without applying
a conductive coating prior to imaging. The enriched nitrifying
culture amended with commercially available Ag NPs (10 nm,
Nanostructured & Amorphous Materials, Inc., Houston, TX)
was placed in an Al cup on a cold stage (10 1C) and imaged at
ca. 7 Torr and 80% relative humidity. In order to obtain higher-
resolution images, bacteria amended with our own Ag NPs
were examined under high-vacuum conditions utilizing a
back-scattered electron (BSE) detector. The nanoparticle
samples synthesized in our laboratory were prepared using
a standard protocol described above, critically point dried,
and coated with 10 nm of Pt.
2.7.
LIVE/DEAD bacterial viability assays
Experiments were carried out in the presence and absence of
nanoparticles to determine the cell viability of heterotrophic
(E. coli PHL628 without GFP tagged) and autotrophic cultures
by using a LIVE/DEAD Baclight
TM
bacterial viability kit
(Molecular Probes, Eugene, OR) (
). Viable and
dead cells were detected by differential staining with a
mixture of a green fluorochrome, SYTO 9 (stains all cells, live
or dead), and a red fluorochrome, propidium iodide (stains
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Table 1 – Composition of the growth nutrients in reactor
influent
Compound
Concentrations in reactor influent
mg/L
Cations
(mM)
Anions
(mM)
Mg(NO
3
)
2
61
0.41Mg
2+
0.82NO
3
Ca(NO
3
)
2
41
0.25Ca
2+
0.25NO
3
NaNO
3
879
10.34Na
+
10.34NO
3
NH
4
NO
3
667
8.33NH
4
+a
8.33NO
3
K
2
HPO
4
3.9
0.04K
+
0.02HPO
4
FeCl
2
4H
2
O
2
0.01Fe
2+
0.02Cl
b
MnSO
4
H
2
O
3.4
0.02 n
2+
0.02SO
4
2c
(NH
4
)
6
Mo
7
O
24
4H
2
O
1.2
0.006NH
4
+a
0.001Mo
7
O
24
6
CuSO
4
0.8
0.01Cu
2+
0.01SO
4
2c
Zn(NO
3
)
2
6H
2
O
1.8
0.01Zn
2+
0.02NO
3
Ni(NO
3
)
2
6H
2
O
0.3
0.001Ni
2+
0.002NO
3
a
Total NH
4
+
¼
8.336 mM.
b
Total Cl
¼
0.02 mM.
c
Total SO
4
2
¼
0.03 mM.
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only bacteria with damaged membranes). A reduction in the
SYTO 9 fluorescent emission results when both dyes are
present in the cell. Dead cells subject to 75% ethanol killing
for 1 h were provided as a positive control. To reduce
background fluorescence, the microbial suspension was
washed with 0.85% NaNO
3
after centrifuging at 10,000g for
15 min. After adding aliquots of microbial suspensions and
stain solution to each well of a 96-well microplate, the plate
was incubated at room temperature in the dark for 15 min,
and the relative fluorescence intensity was measured by the
VICTOR fluorescence microreader. Enumeration of stained
cells was facilitated by excitation at 485 nm and detection at
642 nm (red) and 535 nm (green), for propidium iodide and
SYTO 9, respectively.
3.
Results and discussion
3.1.
Characterization of Ag NPs and AgCl colloids
The absorption spectrum (
) of dark brown Ag NPs
prepared by chemical reduction showed a surface plasmon
absorption band with a maximum of about 400 nm, a
characteristic peak of Ag NPs (
;
), indicating the presence of Ag NPs in the
solution. Due to the excitation of plasma resonances
or interband transitions, some metallic nanoparticle disper-
sions exhibit unique bands/peaks (
). The broadness of the peak is a good indicator of
the size of nanoparticles. As the particle size increases,
the peak becomes narrower with a decreased bandwidth
and an increased band intensity (
). Furthermore, there is an inverse linear
relationship
between
the
full-width
at
half-maxi-
mum (FWHM) and the diameter of particles (
FWHM ¼ 50 þ
230
D
(2)
where both FWHM and the particle diameter (D) are in
nanometers. The size of the Ag NPs was estimated as
approximately 16 nm based on Eq. (2). This result is consistent
with the STEM results, which showed a size distribution
between 10 and 40 nm (
) of the Ag NPs with an average of
14
76 nm.
A shoulder at approximately 425 nm was noticed in UV–vis
absorption spectra, indicating a broad distribution of particle
sizes and shapes in the solution because of crystallization, as
was confirmed by STEM imaging. The position and the
number of peaks in the absorption spectra are dependent
on the shape of the particles: for an ellipsoidal particle there
are two peaks whereas for spherical silver particles there is
only one peak centered at about 400 nm (
The concentrations of Ag
+
ions were measured simulta-
neously to evaluate the stability of Ag NPs in the suspension.
The beginning Ag
+
concentration to make the Ag NP suspen-
sions was 27 mg/L (0.25 mM). At the completion of the
reaction, the residual Ag
+
concentration was 0.6
70.1 mg/L.
The Ag
+
concentration remained largely unchanged at the
end of 1 day of resting at room temperature. Afterward, the
Ag
+
concentrations increased gradually (data not shown), as
also indicated from the changes of solution color.
During a week of Ag
+
monitoring at room temperature, the
color of Ag NP suspensions changed from dark brown to
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0
0.5
1
1.5
2
250
350
450
550
650
Wavelength, (nm)
Absorbance
Fig. 1 – UV–vis absorption spectra of an Ag NP suspension
recorded immediately after chemical reduction (solid line)
and after 1 week (dash line) at room temperature.
0
5
5
10
10
15
20
25
30
35
15
20
25
30
35
Diameter, (nm)
Relative Abundance, (%)
a
b
Fig. 2 – STEM image of Ag NPs prepared by chemical reduction (a) and the particle size distribution (b). The average particle
size was 14 nm. Bar size: 500 nm.
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yellow, presumably due to oxidative dissolution of the Ag NPs
4Ag þ O
2
þ
2H
2
O ¼ 4Ag
þ
þ
4OH
(3)
The color change associated with particle dissolution and
the presence of multiple UV–vis absorption bands indicate
the existence of Ag NPs of various shapes and sizes, as was
confirmed by STEM imaging (
). To minimize the
interference of dynamic changes of Ag NPs, we used the
freshly prepared Ag NP suspensions that were stored shortly
(a few days) at 4 1C before use, during which no significant
changes of Ag
+
concentrations were observed in the suspen-
sion (data not shown).
Silver chloride colloids (100 mg/L Ag) were prepared with an
average size of ca. 0.25 mm. The particle sizes ranged from 0.1
to 2 mm. A constant low Ag
+
concentration was detected
because of the overdose of chloride. The fraction of Ag
+
was
measured to be less than 0.1% of the total Ag in the AgCl
colloidal solution.
3.2.
Effect of Ag species on autotrophic growth
An extant respirometric technique was developed to deter-
mine biokinetic parameters from small pulses of substrate
(e.g., NH
4
+
) while minimizing changes in the microbial
physiological state (
;
).
shows a representative respirograph of ammonia
oxidation after an aliquot of ammonium was injected at
100 s in the enriched nitrifying microbial suspension. The
lack of change in DO illustrates nitrification inhibition in the
presence of Ag NPs. There was no significant pH change
before and after the test because of the addition of MOPS.
As shown in
, at 1 mg/L Ag in the nitrifying suspension,
the inhibitions by Ag NPs, Ag
+
ions, and AgCl colloids were
86
73%, 4277%, and 4674%, respectively. Of all the Ag species
tested, Ag NPs presented the highest inhibition on nitrifying
bacterial growth. Interestingly, the freshly prepared AgCl
colloids with an average size of 0.25 mm also inhibited
nitrification. At the same level of Ag dose, there was no
statistical difference (p40.05) of inhibition between AgCl
colloids and Ag
+
ions. At this small size, silver chloride
colloids appeared to reduce the bacterial growth as effectively
as Ag
+
.
3.3.
Effect of Ag species on heterotrophic growth
Consistent with the results from autotrophic growth study, Ag
NPs inhibited E. Coli growth. While no inhibition was observed
at Ag NP concentrations below 1.0 mM, the heterotrophic
growth rate was reduced significantly by 55% as the Ag NP
concentrations increased to 4.2 mM (
). The IC 50
inhibition by the Ag NP suspension, or the half maximal
inhibitory concentration, was estimated to be 4.0 mM (n ¼ 8).
Surprisingly, silver ion was the most toxic species to inhibit
heterotrophic growth. At 4.2 mM (0.5 mg/L Ag), the inhibi-
tions on the growth of E. coli PHL628-gfp were 55
78%, 100%,
and 66
76% by Ag NPs, Ag
+
ions, and AgCl colloids, respec-
tively. E. Coli treated with 1 mg/L Ag (or 9.3 mM) in the forms of
Ag NPs, Ag
+
ions, or AgCl did not exhibit signs of growth (data
not shown). The inhibition on heterotrophic growth appeared
to be more severe from the long-term microtiter fluorescence
assays, as we reported earlier that inhibition on microbial
growth with longer period of metal exposure tends to be more
significant (
A slight lag phase of E. coli growth (1.5 h) was observed
during the automatic microtiter assays (
). Stationary
phase was reached after incubation of the heterotrophic
strain for approximately 12 h at room temperature (25
71 1C).
Upon the addition of Ag NPs in the microbial suspension, a
slight decrease of fluorescence efficiency (i.e., fluorescence
quenching) with increasing Ag NP concentrations was
observed. In the case of AgCl colloids, the quenching effect
was less significant (data not shown). The results are
consistent with the existing experimental data (
;
), indicating that the
overlap between the GFP-tagged microbial fluorescence and
the plasmon absorption of Ag nanoparticles may slightly
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0
5
10
15
20
25
0
200
400
600
800
Time, (s)
O
2
, (mg/L)
B
A
Fig. 3 – Nitrification inhibition inferred from the decrease of
specific oxygen uptake rate (slope of curve A) in the
presence of Ag NPs, as compared with control (curve B) after
an aliquot of ammonium nitrate was injected individually at
approximately 100 s.
0
20
40
60
80
100
0
0.2
0.4
0.6
0.8
1
Silver concentration, (mg/L)
Inhibition, (%)
Ag NPs
AgCl
Ag
+
Fig. 4 – Nitrification inhibition as a function of the
concentrations of silver in the form of Ag NPs, Ag
+
ions,
and AgCl colloids. Error bars indicate one standard
deviation.
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cause the quenching of the excited-state of GFP molecules on
the Ag NPs.
3.4.
Microscopic observation of microbial/nanoparticle
interaction
The microbial–nanoparticle interaction was visualized by
ESEM, a specialized technique capable of imaging hydrous
samples without the need of pretreatment for conductive
coating (
;
). Commer-
cially available Ag NPs (Nanostructured & Amorphous Mate-
rials, Inc., advertised powder size of 10 nm) aggregated in
water and the nitrifying bacterial suspension. It appeared that
the particles were embedded in microbial extracellular
polymeric substances (
). The true size (from 200 nm to
a few mm) of Ag NPs in water suspension was significantly
different from the claimed size of commercial nanopowders,
consistent with the results reported by others (
).
Higher-resolution electron micrographs were obtained
using BSE mode. After mixing a freshly prepared Ag NP
suspension with the mixed and enriched nitrifying cultures,
it appeared that Ag NPs were adsorbed to the micro-
bial surfaces, probably causing cell wall pitting (
).
Additional work is underway to take higher-resolution images
in order to better understand the microbial–nanoparticle
interactions.
3.5.
Cell membrane integrity inferred from LIVE/DEAD
assays
The fluorescence intensities of the stained microbial cells at
535 nm (green) and 642 nm (red) represent live and dead cells,
respectively. The green/red fluorescence ratio, obtained by
dividing the green and red intensities, was applied to
compare the difference among various treatments by Ag
species. At 1 mg/L Ag, the ratio obtained from the microbial
suspensions treated with Ag NPs showed no significant
difference compared to controls (P40.05), indicating that
there is no evidence of cell membrane leakage caused by Ag
NPs. Similar results were observed in samples treated with
Ag
+
ions or AgCl colloids.
3.6.
Inhibition comparison and mode of antimicrobial
action
Among the Ag species tested, the freshly prepared Ag NPs
presented the highest inhibition to autotrophic nitrifying
organisms. In contrast, silver ion appeared to be most toxic to
heterotrophic growth. Different experimental assays chosen
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Table 2 – Specific growth rates (l) of E. coli PHL628-gfp and the inhibitions by silver species at different concentrations
Concentration (mM)
Ag NP
Ag
+
AgCl colloid
m
(d
1
)
Inhibition (%)
m
(d
1
)
Inhibition (%)
m
(d
1
)
Inhibition (%)
1.4
0.40 (
70.03)
17 (
75)
0.41 (
70.02)
11 (
74)
0.43 (
70.01)
7 (
74)
2.8
0.34 (
70.03)
30 (
76)
0.14 (
70.03)
69 (
76)
0.35 (
70.02)
24 (
75)
4.2
0.22 (
70.04)
55 (
78)
0
100
0.16 (
70.03)
66 (
76)
200
0
5
10
15
20
220
240
260
280
300
320
Time, (h)
RFU, (×1,000)
Fig. 5 – Effect of Ag NP concentrations ( , 0 lM; K, 1.4 lM;
J
,
2.8 lM; ’, 4.2 lM; &, 9.3 lM) on the growth of E. coli PHL628-
gfp, as measured by relative fluorescence units.
Fig. 6 – Silver nanoparticles adsorbed to the enriched
nitrifying culture on a copper grid using ESEM. Arrows
show aggregated AgNPs that attached to microbial cells or
embedded in microbial extracellular polymeric substances.
Bar size: 100 lm.
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for the enriched nitrifying bacteria and heterotrophic E. coli
cells made it difficult to compare the toxicity of Ag NPs to the
two different bacterial species. The difference in toxicity may
be attributed to different growth conditions and cell proper-
ties. The nitrifying bacteria were completely mixed in the
respirometric bottles, aerated with pure oxygen and their
activities were monitored by oxygen uptake rate measure-
ments. Conversely, the E. coli cells were mixed intermittently
in microwells, aerated with ambient air and their activities
were inferred from fluorescence measured over a prolonged
period of time (1 d). Because they have a faster growth rate
than nitrifying bacteria, E. coli cells may have stronger
oxidizing/reducing power and interact with Ag species to
form cell-particle aggregates (
;
), as visible from
, or
produce extracellular or intracellular Ag NPs (
), causing more complex growth problems.
Nitrifying bacteria have remarkably complex internal mem-
brane systems where ammonia monooxygenase (AMO, re-
sponsible for ammonia oxidation to produce hydroxylamine,
NH
2
OH) is located, whereas hydroxylamine oxidoreductase
(HMO) is located in the periplasm (
).
Therefore, we speculate that Ag NPs may have a direct impact
on nitrifying cell membranes where key ammonia oxidation
enzymes are located.
The use of PVA during the synthesis of Ag NPs to control the
nanoparticles size could affect the antibacterial activity by
the coverage of PVA on AgNPs to prevent the direct contact
with bacteria. Due to the significant dilution (1:25 or higher) of
the Ag NP suspension and solvent competition in cell
cultures, the effect of PVA on nanosilver toxicity, however,
would be minimal.
Nanoparticles have substantially different physiochemical
properties from those of bulk materials of the same composi-
tion, possibly resulting in different toxicity mechanisms to
biological systems (
). The mode of antimicro-
bial action by Ag NPs could be the inhibition of the microbial
processes on the cell surface and in the cell. Previous research
demonstrated that Ag NPs attach to the surface of cell
membrane, causing the change of membrane permeability,
dissipation of the ATP pool and proton motive force, and
finally cell death (
;
). The results from our bacterial
viability tests indicated that there is no evidence of the cell
membrane leakage caused by any Ag species at 1 mg/L Ag.
The size of the Ag NPs used in this study was 14
76 nm. These
particles would be too large to diffuse into the cell, as only the
smaller particles mainly in the range of 1–10 nm could enter
the cell based on indirect microscopic evidences (
). Although electrophoresis studies indicated no
direct effect of Ag NPs on intracellular DNA or protein
expression (
), our recent results demon-
strated that inhibition by the Ag NPs might be attributed to
the accumulation of intracellular ROS (
).
Bulk silver toxicity is generally governed by the total
concentration of labile dissolved intracellular Ag species
(
). In the cell, silver ions may deactivate cellular
enzymes and DNA by reacting with electron-donating groups
such as thiol (–SH) groups and generate ROS (
;
). Because of its cationic nature
and its strong association with various ligands in natural
waters, the toxicity of Ag
+
ions depends largely on the
strength and amount of the ligands present (
).
The freshly prepared AgCl colloids can be viewed as one of the
labile species with respect to their small size and low stability
constant (log K
1
¼
3.3) in solution (
).
Depending on their size and bioavailability, the inhibition
caused by AgCl colloids can be as significant as that of Ag
+
ions.
3.7.
Ag NP dissolution
The time-dependent increases of Ag
+
concentrations and
associated color changes of the Ag NP suspension demon-
strated the complexity of various processes such as oxidation,
crystallization, dissolution and aggregation involved in mi-
crobial–nanoparticle interactions. Previous research showed
that Ag NPs were susceptible to oxidation by oxygen, and the
partially oxidized particles appeared more toxic than the
freshly prepared nanoparticles (
). Others found,
however, that the concentration of Ag
+
decreased by 80% after
24 h, possibly due to Ag
0
cluster formation (
). When the Ag NPs were added into a liquid medium, the
antimicrobial effectiveness appeared to decrease when com-
pared to that on the agar plates, presumably because of
microbial-induced coagulation of nanoparticles (
). Experiments involving synthetic zinc
sulfide nanoparticles and representative amino acids also
indicated a driving role of microbially derived extracellular
proteins in rapid nanoparticle aggregation (
). Further research is required to investigate nanoparticle
properties such as size, shape, dissolution/aggregation,
ARTICLE IN PRESS
Fig. 7 – Silver nanoparticles adsorbed to the enriched
nitrifying culture using a high-speed BSE detector. Arrows
show spherical or hexagon types of Ag NPs that attached to
the microbial cells, probably causing cell wall pitting. Bar
size: 500 nm.
W A T E R R E S E A R C H
4 2 ( 2 0 0 8 ) 3 0 6 6 – 3 0 7 4
3072
surface coating, and solubility that may affect the specific
physicochemical and transport properties, which could exert
a significantly different impact on microbial growth (
).
3.8.
Environmental application and implication
The numerous engineered nanomaterials with different sizes,
shapes, compositions, and coatings require high-throughput
benchmarked protocols to screen for potential hazards in the
environment (
). The developed extant
respirometric assay and the automatic microtiter assay
employed in this research are suitable for toxicity assessment
of nanomanterials to microorganisms. The bacteria selected
for each assay, however, are generally not exchangeable
between the two assays. Because of the intrinsic slow
growth rate (about an order of magnitude lower than that
of heterotrophs) of autotrophic nitrifying bacteria and their
high oxygen uptake (4.3 mg O
2
/mg of NH
4
+
-N oxidized to
nitriate) (
),
the
enriched nitrifying
cultures are particularly useful in respirometric assays, but
failed to show significant growth in the cell-enumeration-
based microtiter assay in this study. In contrast, E. coli
cells were easily determined with the automatic microtiter
assay because of their fast growth rate, but failed to pro-
duce meaningful oxygen profiles from the extant respioro-
metric assay because of the low biomass concentrations
from overnight batch cultivation and their low oxygen
uptake
constants
(0.5 mg O
2
/mg COD
removed)
The results of nanosilver toxicity to environmentally
sensitive nitrifying microorganisms suggest that stringent
regulations of Ag NPs entering WWTPs are necessary.
Nitrifying microorganisms involved in nitrification are critical
to biological nutrient removal in modern wastewater treat-
ment. Research is underway to evaluate the fate and impact
of Ag NPs in wastewater treatment systems.
4.
Conclusions
The nature of the cell growth and oxygen uptake behavior
allowed us to determine nanosilver toxicity by applying two
independent microbial growth assays—extant respirometric
assay and automatic microtiter assay—for nitrifying organ-
isms and E. coli cells, respectively. The following conclusions
were drawn from this work:
(1) Silver nanoparticles (Ag NPs) strongly inhibited microbial
growth. Based on a short-term batch respirometric assay,
at 9.3 mM Ag (i.e., 1 mg/L Ag), the inhibitions on nitrifying
bacterial growth by Ag NPs, Ag
+
ions, and AgCl colloids
were 86
73%, 4277%, and 4674%, respectively. Based on a
prolonged microtiter assay, at 4.2 mM Ag, the inhibitions on
the growth of E. coli PHL628-gfp by Ag NPs, Ag
+
ions, and
AgCl colloids were 55
78%, 100%, and 6676%, respectively.
(2) Silver chloride colloids inhibited microbial growth. De-
pending on their particle size and bioavailability, the
inhibition by AgCl colloids can be as significant as that by
Ag
+
ions.
(3) There was no evidence of change in cell membrane
integrity at 1 mg/L Ag for all of the Ag species tested
based on the results from the bacterial LIVE/DEAD assays.
Acknowledgments
This research work was supported by the University of
Missouri Research Board and the National Science Foundation
under Grant no. 0650943. Any opinions, findings, and conclu-
sions or recommendations expressed in this material are
those of the author(s) and do not necessarily reflect the views
of the National Science Foundation.
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Document Outline
- The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth
- Introduction
- Materials and methods
- Results and discussion
- Characterization of Ag NPs and AgCl colloids
- Effect of Ag species on autotrophic growth
- Effect of Ag species on heterotrophic growth
- Microscopic observation of microbial/nanoparticle interaction
- Cell membrane integrity inferred from LIVE/DEAD assays
- Inhibition comparison and mode of antimicrobial action
- Ag NP dissolution
- Environmental application and implication
- Conclusions
- Acknowledgments
- References
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